This invention relates to genetic engineering of yeast, and to the use of certain promoters to improve the results of such genetic engineering.
One of the goals of genetic engineering in yeast is to construct strains of yeast that produce large amounts of a desired protein or enzyme. A particular protein or enzyme may be of interest as a product itself (for example, human alpha interferon), or a protein or enzyme may be of interest because it facilitates production by the host yeast of a desired metabolite (for example, ethanol or an amino acid) or production of a food or beverage product (for example, beer, wine, bread, or spirits). Although it is now routine to obtain some level of expression in yeast of any desired gene into its corresponding protein, the attainment of levels of expression high enough to allow a process to be performed profitably is often difficult.
The term "promoter", as used in connection with the expression of genes in yeast, has a meaning broader than the meaning of the term when used in connection with E. coli, the organism for which promoters were first described. The term "promoter", as used herein, means any DNA sequence which, when associated with a structural gene in a host yeast cell, increases, for that structural gene, one or more of 1) transcription, 2) translation, or 3) mRNA stability, compared to transcription, translation, or mRNA stability in the absence of the promoter sequence, under appropriate growth conditions (e.g., if the promoter is inducible, in the presence of the inducing substance). "mRNA stability" means longer half-life of mRNA.
Several strong promoters are known in the art and have been shown to be useful for expression of heterologous genes in yeast. (A "heterologous gene" means any gene that is not naturally functionally associated with a given promoter, whether that gene is derived from yeast or not.) For example, promoters naturally associated with the Saccharomyces cerevisiae genes TPI1 (triose phosphate isomerase), PGK1 (phosphoglycerate kinase), PYK1 (pyruvate kinase), TDH1, TDH2, and TDH3 (glyceraldehyde phosphate dehydrogenase or triose phosphate dehydrogenase), and ENO1 (enolase 1) have been described as useful for expression of heterologous genes in yeast (Kawasaki, U.S. Pat. No. 4,599,311; Kingsman and Kingsman, U.S. Pat. No. 4,615,974; Burke et al., EPO patent application Ser. No. 84300091.0; and Nunberg et al., WPO patent application Ser. No. 84/02921). All of the above genes encode enzymes which are involved in the glycolytic pathway of yeast, and which are among the most abundant enzymes in the yeast cytoplasm (Brousse et al. (1985) Applied and Environmental Microbiology 50: 951).
Some yeast strains have two different enzymes which act as enolases in the glycolytic pathway. In S. cerevisiae, these two enzymes have been given the names enolase 1 and enolase 2, which are encoded by the ENO1 and ENO2 genes, respectively, each of which has associated with it its distinct promoter sequences (Holland et al. (1980) J. Biol. chem. 257: 7181 and Cohen et al. (1986) Mol. Cell Biol. 6: 2287). In S. cerevisiae, using glucose as a carbon source, enolase 2 is more abundant than enolase 1. As used herein, "enolase 2" refers to the most abundant enolase of any yeast strain using glucose as a carbon source, and "ENO2 promoter" refers to a promoter sequence naturally associated with the gene encoding the most abundant enolase in a yeast strain using glucose as a carbon source.
There have been some attempts to make hybrid yeast promoters. For example, Kingsman et al., EPO 258 067, describe work in which substitution of the PGK UAS with the GAL1,10 UAS confers galactose regulation on the PGK promoter, but insertion of the GAL1,10 UAS at a site upstream or downstream from the PGK UAS, so that both PGK and GAL1,10 UAS's were present in the promoter sequence, did not result in galactose regulation of PGK gene expression.